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1.
Nanoscale ; 13(32): 13746-13757, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34477649

RESUMO

Manipulation of temperature can be used to actuate DNA origami nano-hinges containing gold nanoparticles. We develop a physical model of this system that uses partition function analysis of the interaction between the nano-hinge and nanoparticle to predict the probability that the nano-hinge is open at a given temperature. The model agrees well with experimental data and predicts experimental conditions that allow the actuation temperature of the nano-hinge to be tuned over a range of temperatures from 30 °C to 45 °C. Additionally, the model identifies microscopic interactions that are important to the macroscopic behavior of the system, revealing surprising features of the system. This combination of physical insight and predictive potential is likely to inform future designs that integrate nanoparticles into dynamic DNA origami structures or use strand binding interactions to control dynamic DNA origami behavior. Furthermore, our modeling approach could be expanded to consider the incorporation, stability, and actuation of other types of functional elements or actuation mechanisms integrated into nucleic acid devices.


Assuntos
Ouro , Nanopartículas Metálicas , DNA , Conformação de Ácido Nucleico , Temperatura
2.
J Nutr Biochem ; 99: 108839, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34411715

RESUMO

Omega-3 or n-3 polyunsaturated fatty acids (PUFAs) are widely studied for health benefits that may relate to anti-inflammatory activity. However, mechanisms mediating an anti-inflammatory response to n-3 PUFA intake are not fully understood. Of interest is the emerging role of fatty acids to impact DNA methylation (DNAm) and thereby modulate mediating inflammatory processes. In this pilot study, we investigated the impact of n-3 PUFA intake on DNAm in inflammation-related signaling pathways in peripheral blood mononuclear cells (PBMCs) of women at high risk of breast cancer. PBMCs of women at high risk of breast cancer (n=10) were obtained at baseline and after 6 months of n-3 PUFA (5 g/d EPA+DHA dose arm) intake in a previously reported dose finding trial. DNA methylation of PBMCs was assayed by reduced representation bisulfite sequencing (RRBS) to obtain genome-wide methylation profiles at the single nucleotide level. We examined the impact of n-3 PUFA on genome-wide DNAm and focused upon a set of candidate genes associated with inflammation signaling pathways and breast cancer. We identified 24,842 differentially methylated CpGs (DMCs) in gene promoters of 5507 genes showing significant enrichment for hypermethylation in both the candidate gene and genome-wide analyses. Pathway analysis identified significantly hypermethylated signaling networks after n-3 PUFA treatment, such as the Toll-like Receptor inflammatory pathway. The DNAm pattern in individuals and the response to n-3 PUFA intake are heterogeneous. PBMC DNAm profiling suggests a mechanism whereby n-3 PUFAs may impact inflammatory cascades associated with disease processes including carcinogenesis.

3.
Nucleic Acids Res ; 49(15): 8987-8999, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34358322

RESUMO

Single molecule force spectroscopy is a powerful approach to probe the structure, conformational changes, and kinetic properties of biological and synthetic macromolecules. However, common approaches to apply forces to biomolecules require expensive and cumbersome equipment and relatively large probes such as beads or cantilevers, which limits their use for many environments and makes integrating with other methods challenging. Furthermore, existing methods have key limitations such as an inability to apply compressive forces on single molecules. We report a nanoscale DNA force spectrometer (nDFS), which is based on a DNA origami hinge with tunable mechanical and dynamic properties. The angular free energy landscape of the nDFS can be engineered across a wide range through substitution of less than 5% of the strand components. We further incorporate a removable strut that enables reversible toggling of the nDFS between open and closed states to allow for actuated application of tensile and compressive forces. We demonstrate the ability to apply compressive forces by inducing a large bend in a 249bp DNA molecule, and tensile forces by inducing DNA unwrapping of a nucleosome sample. These results establish a versatile tool for force spectroscopy and robust methods for designing nanoscale mechanical devices with tunable force application.


Assuntos
DNA/química , Nanoestruturas/química , Bioengenharia , Fenômenos Biomecânicos , Nucleossomos/química , Análise Espectral
4.
Sci Rep ; 11(1): 14126, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34238982

RESUMO

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. The molecular characteristics of histologically normal appearing tissue adjacent to the tumor (NAT) from PTC patients are not well characterized. The aim of this study was to characterize the global gene expression profile of NAT and compare it with those of normal and tumor thyroid tissues. We performed total RNA sequencing with fresh frozen thyroid tissues from a cohort of three categories of samples including NAT, normal thyroid (N), and PTC tumor (T). Transcriptome analysis shows that NAT presents a unique gene expression profile, which was not associated with sex or the presence of lymphocytic thyroiditis. Among the differentially expressed genes (DEGs) of NAT vs N, 256 coding genes and 5 noncoding genes have been reported as cancer genes involved in cell proliferation, apoptosis, and/or tumorigenesis. Bioinformatics analysis with Ingenuity Pathway Analysis software revealed that "Cancer, Organismal Injury and Abnormalities, Cellular Response to Therapeutics, and Cellular Movement" were major dysregulated pathways in the NAT tissues. This study provides improved insight into the complexity of gene expression changes in the thyroid glands of patients with PTC.

5.
Phys Rev E ; 103(5-1): 052404, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34134264

RESUMO

Protein complexes involved in DNA mismatch repair diffuse along dsDNA as sliding clamps in order to locate a hemimethylated incision site. They have been observed to use a dissociative mechanism, in which two proteins, while continuously remaining attached to the DNA, sometimes associate into a single complex sliding on the DNA and sometimes dissociate into two independently sliding proteins. Here, we study the probability that these complexes locate a given target site via a semi-analytic, Monte Carlo calculation that tracks the association and dissociation of the sliding complexes. We compare such probabilities to those obtained using a nondissociative diffusive scan in the space of physically realistic diffusion constants, hemimethylated site distances, and total search times to determine the regions in which dissociative searching is more or less efficient than nondissociative searching. We conclude that the dissociative search mechanism is advantageous in the majority of the physically realistic parameter space, suggesting that the dissociative search mechanism confers an evolutionary advantage.

6.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495337

RESUMO

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.


Assuntos
Doxorrubicina/efeitos adversos , Traumatismos Cardíacos/induzido quimicamente , Traumatismos Cardíacos/tratamento farmacológico , Terapia de Alvo Molecular , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Animais , Criança , Regulação da Expressão Gênica , Traumatismos Cardíacos/fisiopatologia , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/deficiência , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Análise de Sequência de RNA
7.
Nucleic Acids Res ; 49(1): 547-567, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330920

RESUMO

Genomic studies have indicated that certain bacterial lineages such as the Bacteroidetes lack Shine-Dalgarno (SD) sequences, and yet with few exceptions ribosomes of these organisms carry the canonical anti-SD (ASD) sequence. Here, we show that ribosomes purified from Flavobacterium johnsoniae, a representative of the Bacteroidetes, fail to recognize the SD sequence of mRNA in vitro. A cryo-electron microscopy structure of the complete 70S ribosome from F. johnsoniae at 2.8 Å resolution reveals that the ASD is sequestered by ribosomal proteins bS21, bS18 and bS6, explaining the basis of ASD inhibition. The structure also uncovers a novel ribosomal protein-bL38. Remarkably, in F. johnsoniae and many other Flavobacteriia, the gene encoding bS21 contains a strong SD, unlike virtually all other genes. A subset of Flavobacteriia have an alternative ASD, and in these organisms the fully complementary sequence lies upstream of the bS21 gene, indicative of natural covariation. In other Bacteroidetes classes, strong SDs are frequently found upstream of the genes for bS21 and/or bS18. We propose that these SDs are used as regulatory elements, enabling bS21 and bS18 to translationally control their own production.


Assuntos
Bacteroidetes/genética , Iniciação Traducional da Cadeia Peptídica , Sequências Reguladoras de Ácido Ribonucleico , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Códon de Iniciação , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/genética , Flavobacterium/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Puromicina/farmacologia , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , RNA Ribossômico 5S/genética , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência , Especificidade da Espécie
8.
J Mol Biol ; 432(19): 5259-5272, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32710983

RESUMO

BipA is a conserved translational GTPase of bacteria recently implicated in ribosome biogenesis. Here we show that Escherichia coli ΔbipA cells grown at suboptimal temperature accumulate immature large subunit particles missing several proteins. These include L17 and L17-dependent binders, suggesting that structural block 3 of the subunit folds late in the assembly process. Parallel analysis of the control strain revealed accumulation of nearly identical intermediates, albeit at lower levels, suggesting qualitatively similar routes of assembly. This came as a surprise, because earlier analogous studies of wild-type E. coli showed early binding of L17. Further investigation showed that the main path of 50S assembly differs depending on conditions of growth. Either supplementation of the media with lysine and arginine or suboptimal temperature appears to delay block 3 folding, demonstrating the flexible nature of the assembly process. We also show that the variant BipA-H78A fails to rescue phenotypes of the ΔbipA strain, indicating a critical role for GTP hydrolysis in BipA function. In fact, BipA-H78A confers a dominant negative phenotype in wild-type cells. Controlled production of BipA-H78A causes accumulation of 70S monosomes at the expense of polysomes, suggesting that the growth defect stems from a shutdown of translation.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato/metabolismo , Hidrólise , Modelos Moleculares , Mutação , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Bactérias/genética
9.
PLoS Genet ; 16(6): e1008830, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502192

RESUMO

Many post-transcriptional mechanisms operate via mRNA 3'UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3'UTR introns (3'UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3'UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3'UI in zebrafish, mouse and humans. foxo3b is one such proximal 3'UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3'UIs may serve gene regulatory function in vertebrates.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Códon de Terminação , Conjuntos de Dados como Assunto , Embrião não Mamífero , Éxons/genética , Redes Reguladoras de Genes/genética , Homozigoto , Humanos , Íntrons/genética , Camundongos , Músculo Esquelético/inervação , Mutagênese , Mutação , Crescimento Neuronal/genética , Proteínas Nucleares/genética , Terminação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Alinhamento de Sequência , Regulação para Cima , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
10.
RNA ; 26(9): 1216-1233, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467309

RESUMO

In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion.


Assuntos
Sítios de Ligação/genética , Ligação Proteica/genética , RNA/química , RNA/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Linhagem Celular , Células Eucarióticas/metabolismo , Éxons/genética , Células HEK293 , Humanos , Imunoprecipitação/métodos , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética
11.
PLoS Comput Biol ; 16(5): e1007852, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32379750

RESUMO

Single nucleotide polymorphisms are widely associated with disease, but the ways in which they cause altered phenotypes are often unclear, especially when they appear in non-coding regions. One way in which non-coding polymorphisms could cause disease is by affecting crucial RNA-protein interactions. While it is clear that changing a protein binding motif will alter protein binding, it has been shown that single nucleotide polymorphisms can affect RNA secondary structure, and here we show that single nucleotide polymorphisms can affect RNA-protein interactions from outside binding motifs through altered RNA secondary structure. By using a modified version of the Vienna Package and PAR-CLIP data for HuR (ELAVL1) in humans we characterize the genome-wide effect of single nucleotide polymorphisms on HuR binding and show that they can have a many-fold effect on the affinity of HuR binding to RNA transcripts from tens of bases away. We also find some evidence that the effect of single nucleotide polymorphisms on protein binding might be under selection, with the non-reference alleles tending to make it harder for a protein to bind.


Assuntos
Conformação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Genoma Humano , Humanos , Ligação Proteica , RNA Mensageiro/química
12.
J Transl Med ; 18(1): 63, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32039730

RESUMO

BACKGROUND: Direct cDNA preamplification protocols developed for single-cell RNA-seq have enabled transcriptome profiling of precious clinical samples and rare cell populations without the need for sample pooling or RNA extraction. We term the use of single-cell chemistries for sequencing low numbers of cells limiting-cell RNA-seq (lcRNA-seq). Currently, there is no customized algorithm to select robust/low-noise transcripts from lcRNA-seq data for between-group comparisons. METHODS: Herein, we present CLEAR, a workflow that identifies reliably quantifiable transcripts in lcRNA-seq data for differentially expressed genes (DEG) analysis. Total RNA obtained from primary chronic lymphocytic leukemia (CLL) CD5+ and CD5- cells were used to develop the CLEAR algorithm. Once established, the performance of CLEAR was evaluated with FACS-sorted cells enriched from mouse Dentate Gyrus (DG). RESULTS: When using CLEAR transcripts vs. using all transcripts in CLL samples, downstream analyses revealed a higher proportion of shared transcripts across three input amounts and improved principal component analysis (PCA) separation of the two cell types. In mouse DG samples, CLEAR identifies noisy transcripts and their removal improves PCA separation of the anticipated cell populations. In addition, CLEAR was applied to two publicly-available datasets to demonstrate its utility in lcRNA-seq data from other institutions. If imputation is applied to limit the effect of missing data points, CLEAR can also be used in large clinical trials and in single cell studies. CONCLUSIONS: lcRNA-seq coupled with CLEAR is widely used in our institution for profiling immune cells (circulating or tissue-infiltrating) for its transcript preservation characteristics. CLEAR fills an important niche in pre-processing lcRNA-seq data to facilitate transcriptome profiling and DEG analysis. We demonstrate the utility of CLEAR in analyzing rare cell populations in clinical samples and in murine neural DG region without sample pooling.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Animais , Camundongos , RNA-Seq , Análise de Sequência de RNA , Transcriptoma/genética , Sequenciamento Completo do Exoma
13.
Nucleic Acids Res ; 48(7): 3806-3815, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-31996904

RESUMO

Cap homeostasis is the cyclical process of decapping and recapping that maintains the translation and stability of a subset of the transcriptome. Previous work showed levels of some recapping targets decline following transient expression of an inactive form of RNMT (ΔN-RNMT), likely due to degradation of mRNAs with improperly methylated caps. The current study examined transcriptome-wide changes following inhibition of cytoplasmic cap methylation. This identified mRNAs with 5'-terminal oligopyrimidine (TOP) sequences as the largest single class of recapping targets. Cap end mapping of several TOP mRNAs identified recapping events at native 5' ends and downstream of the TOP sequence of EIF3K and EIF3D. This provides the first direct evidence for downstream recapping. Inhibition of cytoplasmic cap methylation was also associated with mRNA abundance increases for a number of transcription, splicing, and 3' processing factors. Previous work suggested a role for alternative polyadenylation in target selection, but this proved not to be the case. However, inhibition of cytoplasmic cap methylation resulted in a shift of upstream polyadenylation sites to annotated 3' ends. Together, these results solidify cap homeostasis as a fundamental process of gene expression control and show cytoplasmic recapping can impact regulatory elements present at the ends of mRNA molecules.


Assuntos
Sequência de Oligopirimidina na Região 5' Terminal do RNA , Capuzes de RNA/metabolismo , RNA Mensageiro/química , Sequências Reguladoras de Ácido Ribonucleico , Linhagem Celular Tumoral , Citoplasma , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Metilação , Poliadenilação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Biophys J ; 117(11): 2204-2216, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31732143

RESUMO

In eukaryotic cells, DNA is packaged into chromatin where nucleosomes are the basic packaging unit. Important cellular processes including gene expression, DNA replication, and DNA repair require nucleosomal DNA to be unwrapped so that functional proteins can access their target sites, which otherwise are sterically occluded. A key question in this process is what the unwrapped conformations individual nucleosomes adopt within chromatin are. Here, we develop a concurrent nucleosome unwrapping model to address this question. We hypothesize that for a given end-to-end distance of the nucleosomal DNA, the nucleosomal DNA stochastically unwraps from the histone core from both ends independently and that this combination of unwrapping from both sides results in a significant increase in the average distance between the DNA extending from both sides of the nucleosomes. We test our model on recently published experiments using a DNA origami nanocaliper that quantifies nucleosome unwrapping and achieve good agreement between experiment and model prediction. We then investigate the DNA origami caliper distribution when attached to a hexasome (a nucleosome lacking an H2A/H2B dimer). A significant shift in the caliper angle distribution caused by the asymmetric structural features of the hexasome seen experimentally is consistent with the model. Our modeling approach may be more broadly useful to the interpretation of other studies of nucleosome dynamics, chromatin dynamics, and regulatory processes involving nucleosome unwrapping, as well as more generally to optimization of future DNA origami designs to probe mechanical properties of biomolecules.


Assuntos
Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Bases , DNA/química , DNA/genética , DNA/metabolismo , Nanoestruturas/química , Termodinâmica
15.
Nucleic Acids Res ; 47(20): 10477-10488, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31602466

RESUMO

In all cells, initiation of translation is tuned by intrinsic features of the mRNA. Here, we analyze translation in Flavobacterium johnsoniae, a representative of the Bacteroidetes. Members of this phylum naturally lack Shine-Dalgarno (SD) sequences in their mRNA, and yet their ribosomes retain the conserved anti-SD sequence. Translation initiation is tuned by mRNA secondary structure and by the identities of several key nucleotides upstream of the start codon. Positive determinants include adenine at position -3, reminiscent of the Kozak sequence of Eukarya. Comparative analysis of Escherichia coli reveals use of the same Kozak-like sequence to enhance initiation, suggesting an ancient and widespread mechanism. Elimination of contacts between A-3 and the conserved ß-hairpin of ribosomal protein uS7 fails to diminish the contribution of A-3 to initiation, suggesting an indirect mode of recognition. Also, we find that, in the Bacteroidetes, the trinucleotide AUG is underrepresented in the vicinity of the start codon, which presumably helps compensate for the absence of SD sequences in these organisms.


Assuntos
Flavobacterium/genética , Regulação Bacteriana da Expressão Gênica , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/química , Proteínas de Bactérias/biossíntese , Flavobacterium/metabolismo , Motivos de Nucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
BMC Bioinformatics ; 20(1): 431, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426747

RESUMO

BACKGROUND: Protein pulldown using Methyl-CpG binding domain (MBD) proteins followed by high-throughput sequencing is a common method to determine DNA methylation. Algorithms have been developed to estimate absolute methylation level from read coverage generated by affinity enrichment-based techniques, but the most accurate one for MBD-seq data requires additional data from an SssI-treated Control experiment. RESULTS: Using our previous characterizations of Methyl-CpG/MBD2 binding in the context of an MBD pulldown experiment, we build a model of expected MBD pulldown reads as drawn from SssI-treated DNA. We use the program BayMeth to evaluate the effectiveness of this model by substituting calculated SssI Control data for the observed SssI Control data. By comparing methylation predictions against those from an RRBS data set, we find that BayMeth run with our modeled SssI Control data performs better than BayMeth run with observed SssI Control data, on both 100 bp and 10 bp windows. Adapting the model to an external data set solely by changing the average fragment length, our calculated data still informs the BayMeth program to a similar level as observed data in predicting methylation state on a pulldown data set with matching WGBS estimates. CONCLUSION: In both internal and external MBD pulldown data sets tested in this study, BayMeth used with our modeled pulldown coverage performs better than BayMeth run without the inclusion of any estimate of SssI Control pulldown, and is comparable to - and in some cases better than - using observed SssI Control data with the BayMeth program. Thus, our MBD pulldown alignment model can improve methylation predictions without the need to perform additional control experiments.


Assuntos
Biologia Computacional/métodos , Metilação de DNA/genética , DNA-Citosina Metilases/metabolismo , DNA/metabolismo , Modelos Biológicos , Alinhamento de Sequência , Algoritmos , Pareamento de Bases , Cromossomos Humanos Par 7/genética , Ilhas de CpG/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Domínio de Ligação a CpG Metilada , Análise de Sequência de DNA/métodos
17.
Clin Cancer Res ; 25(20): 6260-6273, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31296529

RESUMO

PURPOSE: Aberrant Myc expression is a major factor in the pathogenesis of aggressive lymphoma, and these lymphomas, while clinically heterogeneous, often are resistant to currently available treatments and have poor survival. Myc expression can also be seen in aggressive lymphomas that are observed in the context of CLL, and we sought to develop a mouse model that could be used to study therapeutic strategies for aggressive lymphoma in the context of CLL. EXPERIMENTAL DESIGN: We crossed the Eµ-TCL1 mouse model with the Eµ-Myc mouse model to investigate the clinical phenotype associated with B-cell-restricted expression of these oncogenes. The resulting malignancy was then extensively characterized, from both a clinical and biologic perspective. RESULTS: Eµ-TCL1xMyc mice uniformly developed highly aggressive lymphoid disease with histologically, immunophenotypically, and molecularly distinct concurrent CLL and B-cell lymphoma, leading to a significantly reduced lifespan. Injection of cells from diseased Eµ-TCL1xMyc into WT mice established a disease similar to that in the double-transgenic mice. Both Eµ-TCL1xMyc mice and mice with disease after adoptive transfer failed to respond to ibrutinib. Effective and durable disease control was, however, observed by selective inhibition of nuclear export protein exportin-1 (XPO1) using a compound currently in clinical development for relapsed/refractory malignancies, including CLL and lymphoma. CONCLUSIONS: The Eµ-TCL1xMyc mouse is a new preclinical tool for testing experimental drugs for aggressive B-cell lymphoma, including in the context of CLL.


Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Células B/genética , Neoplasias Primárias Múltiplas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas/genética , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Carioferinas/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Primárias Múltiplas/patologia , Estudo de Prova de Conceito , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Células Tumorais Cultivadas/transplante
18.
Blood ; 134(5): 432-444, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31151986

RESUMO

Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eµ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/antagonistas & inibidores , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Metilação de DNA , Modelos Animais de Doenças , Epigênese Genética , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/química , Nanopartículas/administração & dosagem , Nanopartículas/química , Taxa de Sobrevida , Nanomedicina Teranóstica , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Nucleic Acids Res ; 47(11): 5617-5633, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31216039

RESUMO

Nucleosomes, the fundamental organizing units of eukaryotic genomes, contain ∼146 base pairs of DNA wrapped around a histone H3-H4 tetramer and two histone H2A-H2B dimers. Converting nucleosomes into hexasomes by removal of a H2A-H2B dimer is an important regulatory event, but its regulation and functional consequences are not well-understood. To investigate the influence of hexasomes on DNA accessibility, we used the property of the Widom-601 Nucleosome Positioning Sequence (NPS) to form homogeneously oriented hexasomes in vitro. We find that DNA accessibility to transcription factors (TF) on the hexasome H2A-H2B distal side is identical to naked DNA, while the accessibility on the H2A-H2B proximal side is reduced by 2-fold, which is due to a 2-fold reduction in hexasome unwrapping probability. We then determined that a 23 bp region of the Widom-601 NPS is responsible for forming homogeneously oriented hexasomes. Analysis of published ChIP-exo data of hexasome containing genes identified two DNA sequence motifs that correlate with hexasome orientation in vivo, while ExoIII mapping studies of these sequences revealed they generate homogeneously oriented hexasomes in vitro. These results indicate that hexasome orientation, which is influenced by the underlying DNA sequence in vivo, is important for modulating DNA accessibility to regulate transcription.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/química , DNA/química , Histonas/química , Nucleossomos/química , Fatores de Transcrição/química , DNA/genética , Regulação da Expressão Gênica , Nucleossomos/genética , Ligação Proteica , Multimerização Proteica , Transcrição Genética
20.
Phys Rev E ; 99(2-1): 022415, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30934369

RESUMO

RNA forms elaborate secondary structures through intramolecular base pairing. These structures perform critical biological functions within each cell. Due to the availability of a polynomial algorithm to calculate the partition function over these structures, they are also a suitable system for the statistical physics of disordered systems. In this model, below the denaturation temperature, random RNA secondary structures exist in one of two phases: a strongly disordered, low-temperature glass phase and a weakly disordered, high-temperature molten phase. The probability of two bases to pair decays with their distance with an exponent 3/2 in the molten phase and about 4/3 in the glass phase. Inspired by previous results from a renormalized field theory of the glass transition separating the two phases, we numerically study this transition. We introduce distinct order parameters for each phase that both vanish at the critical point. We finally explore the driving mechanism behind this transition.


Assuntos
Vidro/química , Conformação de Ácido Nucleico , Transição de Fase , RNA/química , Sequência de Bases , Modelos Moleculares , RNA/genética , Temperatura de Transição
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